Reciprocal Transplantation of the Heterotrophic Coral Tubastraea

Received: 21 August 2019 | Revised: 6 December 2019 | Accepted: 9 December 2019 DOI: 10.1002/ece3.5959

ORIGINAL RESEARCH

Reciprocal transplantation of the heterotrophic coral Tubastraea coccinea (Scleractinia: Dendrophylliidae) between distinct habitats did not alter its venom toxin composition

1Departamento de Ciências do Mar, Universidade Federal de São Paulo, Santos, Abstract Brazil Tubastraea coccinea is an azooxanthellate coral species recorded in the Indian and 2 Centro de Biologia Marinha (CEBIMar), Atlantic oceans and is presently widespread in the southwestern Atlantic with an Universidade de São Paulo, São Sebastião, Brazil alien status for Brazil. T. coccinea outcompete other native coral species by using 3Departamento de Zoologia, Instituto de a varied repertoire of biological traits. For example, T. coccinea has evolved potent Biociências, Universidade de São Paulo, São Paulo, Brazil venom capable of immobilizing and digesting zooplankton prey. Diversification and 4Departmento de Microbiologia, Instituto de modification of venom toxins can provide potential adaptive benefits to individual Ciências Biomédicas, Universidade de São fitness, yet acquired alteration of venom composition in cnidarians is poorly under- Paulo, São Paulo, Brazil stood as the adaptive flexibility affecting toxin composition in these ancient lineages 5School of Cancer & Pharmaceutical Sciences, Faculty of Life Sciences & has been largely ignored. We used quantitative high-throughput proteomics to de- Medicine, King's College London, London, tect changes in toxin expression in clonal fragments of specimens collected and inter- UK 6Aulesa Biosciences Ltd, Shefford, UK changed from two environmentally distinct and geographically separate study sites. 7Faculdade de Ciências Farmacêuticas, Unexpectedly, despite global changes in protein expression, there were no changes in Universidade de São Paulo, São Paulo, Brazil the composition and abundance of toxins from coral fragments recovered from either

Correspondence site, and following clonal transplantation between sites. There were also no apparent Paul F. Long, School of Cancer & changes to the cnidome (cnidae) and gross skeletal or soft tissue morphologies of the Pharmaceutical Sciences, Faculty of Life Sciences & Medicine, King's College London, specimens. These results suggest that the conserved toxin complexity of T. coccinea UK. co-evolved with innovation of the venom delivery system, and its morphological de- Email: [email protected] velopment and phenotypic expression are not modulated by habitat pressures over Funding information short periods of time. The adaptive response of the venom trait to specific predatory Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Grant/Award regimes, however, necessitates further consideration. Number: 001; Fundação de Amparo à Pesquisa do Estado de São Paulo, Grant/ KEYWORDS Award Number: 2011/50242-5 and 2014/01332-0; Universidade de São Paulo, cnidaria, fitness, proteomics, reciprocal transplantation, toxin diversification, venom Grant/Award Number: 13.1.1502.9.8; Conselho Nacional de Desenvolvimento Científico e Tecnológico, Grant/Award Number: 301436/2018-5 and 309995/ 2017-5; Leverhulme Trust, Grant/Award Number: RPG-2016-037

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd

.www.ecolevol.org Ecology and Evolution. 2020;10:1794–1803 | 1794 KITAHARA et al. | 1795

1 | INTRODUCTION Corals of the genus Tubastraea are obligate heterotrophs, which lack autotrophic dinoflagellate endosymbionts to provide host photosyn- The major and most lethal components of animal venoms are protein thetic nutritional energy, so instead feed predominantly on pelagic and peptide toxins. The toxin compositions of venom are attributed zooplankton. T. coccinea is considered an alien and invasive species to serve in both offensive and defensive functions to facilitate prey to the southwestern Atlantic Ocean that has now expanded its habi- capture and provide protection from predation (Casewell, Wüster, tat range to much of Brazil's southern coastal reefs, which is believed Vonk, Harrison, & Fry, 2013). Animals must adjust to divergent and to have been propagated by a single invasion event in the 1990s (re- changing conditions in their biotic and abiotic environment, which viewed by Miranda, Costa, Lorders, Nunes, & Barros, 2016). T. coc- offer opportunities or pose challenges for feeding and defense. cinea is a hermaphroditic coral; therefore, settlement of broadcast Adapting toxin composition to accommodate these changes has planula via sexual reproduction does not occur. Proliferation ensues been extensively documented in prominent venomous bilaterians. by the release of brooding larvae (Ayre & Resing, 1986) with high For example, variability in snake venom toxins has been associated fecundity (de Paula, Pires, & Creed, 2014), or is spread by runners with geographical distribution (Alape-Girón et al., 2008) and their (Vermeij, 2005) and by the fragmentation of colonies, including ecological conditions (Strickland et al., 2018), but are most widely at- regeneration from undifferentiated coral tissue which fosters the tributed with their ability to capture, consume, and digest a wide va- rapid propagation of clonal offspring (Capel, Migotto, Zilberberg, & riety of different prey types (Daltry, Wüster, & Thorpe, 1996; Gibbs Kitahara, 2014; Luz et al., 2018). & Mackessy, 2009). Likewise, geographical variations in venom toxin Recent proteomic analysis of nematocyst venom isolated from composition have been documented in some scorpions, spiders, and primary tentacles of T. coccinea revealed a complement of 17 likely species of cone snails, which has been linked also to changes in hab- toxins, mainly with predicted cytolytic or protease inhibitor activi- itat or diet (Abdel-Rahman, Omran, Abdel-Nabi, Ueda, & McVean, ties, consistent with consuming a zooplankton diet (Jaimes-Becerra 2009; Duda, Chang, Lewis, & Lee, 2009; Pekár, Petráková, Šedo, et al., 2019). Given the vastly different trophic and interspecific in- Korenko, & Zdráhal, 2018). However, data are scarce or nonexistent teractions cnidarians encounter in diverse benthic and pelagic hab- for geographical or intraspecific variation for the majority of the itats, surprisingly little is known about their venom composition in venomous taxa. response to diverse ecological conditions. Here, we use a tandem Cnidaria (corals, sea anemones, hydroids, jellyfish, myxozoans, mass tag (TMT)-based proteomics approach to compare wild clonal and kin) possess a unique venom delivery system–nematocytes or populations of T. coccinea from near-shore and offshore habitats to “stinging cells,” that release their toxic payload by discharging a pen- determine whether geographical distribution and environmental etrative barb from an intracellular cnida organelle called a nemato- factors could influence venom toxin modification in this early diverg- cyst. Approximately 30 different varieties of nematocyst are known ing metazoan, or whether such alterations are elaborated only by to exist, but individual species usually combine no more than two more advanced bilaterian taxa. to six structural types that are collectively known as the organism's cnidome (Östman, 2000). Cnidarians are possibly the earliest diverg- ing venomous animal lineage to deploy venom for both predation 2 | MATERIALS AND METHODS and defense (Jaimes-Becerra et al., 2017). Venom production and maintenance are therefore central to cnidarian existence and evolu- 2.1 | Study sites and sampling design tion, and increasingly more is known about the evolutionary history and phyletic distribution of cnidarian toxins. A pattern is emerging Clonal populations of T. coccinea from two environmentally dis- which suggests venoms with predominantly cytolytic and neurotoxic tinct and geographically separate study sites were collected from activities were established by early cnidarian ancestors followed by the northern coast of São Paulo State, Brazil, and were analyzed for lineage-specific recruitment of certain toxin protein families, with changes in protein expression at the beginning and end of a six-week cytolysins diversifying prominently in Medusozoa and neurotoxins period of transplantation (Figure 2). All field work was conducted in Anthozoa (Balasubramanian et al., 2012; Brinkman et al., 2015; with appropriate permissions (MAA Research permit number 36717- Huang et al., 2016; Jaimes-Becerra et al., 2017; Li et al., 2014, 2016; 17). Reciprocal transplantations were made between an offshore Macrander, Brugler, & Daly, 2015; Madio, Undheim, & King, 2017; site (Ilha dos Búzios; −23.81434, −45.20562) and an inshore site Ponce, Brinkman, Potriquet, & Mulvenna, 2016; Rachamim et al., (Ilha Bella yacht club; −23.813611, −45.369699). The offshore and 2015). When an innovative unsupervised clustering approach was inshore sites are 40 km apart and separated by São Sebastião Island used to compare toxin composition between groups of venomous (Ilhabela municipality) and São Sebastião Channel. The offshore site animals, the results revealed that despite the early divergence and has notably greater sea surface current speeds, lower temperature, morphological simplicity of cnidarians, their toxin composition was and lower turbidity regimes compared to that of the inshore habi- as complex as those of venomous insects, gastropods, and elapid tat site, but otherwise the two locations were similar in depth. In snakes (Jaimes-Becerra et al., 2019). September 2017, large (approx.) 15 × 15 cm colonies of T. coccinea The orange cup coral Tubastraea coccinea Lesson 1829 (Figure 1) were collected from wild populations from each site and sectioned is presently broadly distributed in the Indian and Atlantic oceans. into nearly equal thirds. One third of each colony was replaced in 1796 | KITAHARA et al.

tissue. The reconstituted material was disrupted on ice using a Qiagen TissueLyser II operated at 20–30 Hz for 2 min. The extracts were then centrifuged for 5 min at 10,000 × g and 4°C. The super- natants were decanted, and soluble protein concentrations were quantified by Nanodrop (Thermo Scientific) spectrophotometry and then lyophilized.

2.4 | Protein preparation and labeling

The lyophilized samples were reconstituted in 50 µl TEAB and 1 µl of each protein extract (equivalent to 25 µg of protein in each sample), protein disulfides were reduced with 10 µl of 8 mM TCEP in 100 mM TEAB and 0.1% (w/v) SDS at 55°C for 1 hr, and then alkylated with 10 µl of 67.5 mM iodoacetamide in 100 mM TEAB and 0.1% (w/v) SDS at room temperature for 30 min in the dark. Trypsin digestion

FIGURE 1 Gross skeletal and soft tissue morphology of was then performed overnight at 37°C by adding 10 µl of 0.2 mg/ Tubastraea coccinea ml trypsin in 100 mM TEAB and 0.1% (v/v) TFA. Following trypsin digestion, each sample was labeled with an isobaric tandem mass tag its native habitat (designated offshore O-T6 and inshore I-T6), while (TMT) 6-plex reagent set using the protocol supplied with the rea- a second fragment was transplanted to the alternate study site gent kit (Lot# SF251226 Thermo Fisher). Once labeled with a unique (Figure 2, designated offshore to inshore OI-Tx and inshore to off- TMT reagent, the six individual samples were combined to create the shore IO-Tx). The third fragment of each colony (designated offshore TMT6plex analytical sample mixture. This combined sample of TMT O-T0 and inshore I-T0) was taken to the Centro de Biologia Marinha labeled peptides was then fractionated into 12 fractions by off-gel (CEBIMar) and stored at −80°C. At completion of the field experi- electrophoresis using an IPG strip pH 3–10 (Bio-Rad) for 20 kV hr in ment, T. coccinea fragments from original and transplanted offshore 1× OFFGEL buffer, pH 3–10. Fractions were solubilized in 50 mM and inshore sites were recovered and stored also at −80°C for sub- ammonium bicarbonate prior to LC-MS/MS. sequent proteomics analyses. The gross morphological features of each coral fragment were photographed at the beginning and end of the transplant experiment. 2.5 | LC-MS/MS tandem mass spectrometry

Chromatographic separations were performed on each fraction 2.2 | Isolation and identification of nematocysts using an Ultimate 3000 nano-LC system in line with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). In brief, pep- Individual T. coccinea polyps are relatively large, and although the tide samples in 1% (v/v) formic acid were injected onto an Acclaim tentacles retract upon freezing, the soft tentacle tissues could easily PepMap C18 nano-trap column (Thermo Scientific). After washing be dissected using forceps and a scalpel blade. Intact nematocysts with 0.5% (v/v) acetonitrile and 0.1% (v/v) formic acid, peptides were isolated from the dissected tentacles as previously described were resolved on a 250 mm × 75 μm Acclaim PepMap C18 reverse- (Weston et al., 2013) and, after microscopic inspection, were lyophi- phase analytical column (Thermo Scientific) over a 150- min organic lized. An optical microscope (Nikon ECLIPSE 80i with a 100x objec- gradient, using 7 gradient segments (1%–6% solvent B over 1 min, tive lens with immersion oil) equipped with a digital camera (Nikon 6%–15% B over 58 min, 15%–32% B over 58 min, 32%–40% B over DS-Ri1) for documentation was used for the identification of nema- 5 min, 40%–90% B over 1 min, held at 90% B for 6 min, and then tocysts. The abundance of different nematocyst types was recorded reduced to 1% B over 1 min.) with a flow rate of 300 nl/min. Solvent as either “very common,” “common,” or “rare” based on the scheme A was 0.1% (v/v) formic acid, and Solvent B was aqueous 80% (v/v) of Picciani, Pires, and Silva (2011). acetonitrile in 0.1% (v/v) formic acid. Peptides were ionized by nano- electrospray ionization at 2.2 kV using a stainless-steel emitter with an internal diameter of 30 μm (Thermo Scientific) and a capillary 2.3 | Protein extraction temperature of 250°C. All spectra were acquired using an Orbitrap Fusion Tribrid mass spectrometer controlled by Xcalibur 2.0 soft- A volume of 200 µl of lysis buffer (8 M urea, 75 mM NaCl, 50 mM ware (Thermo Scientific) and operated in data-dependent acquisi- Tris, pH 8.2, plus one tablet each of Roche protease (cOmplete) and tion mode. FTMS1 spectra were collected at a resolution of 120,000 phosphatase (PhosStop) inhibitor cocktail per 10 ml of lysis buffer) over a scan range (m/z) of 350–1,550, with an automatic gain control was added to approximately 10 mg of freeze dried nematocyst (AGC) target of 400,000 and a maximum injection time of 100 ms. KITAHARA et al. | 1797

I-T0 O-T0

I-T6 O-T6

IO-Tx I-T0 O-T0 I-T6 O-T6 OI-Tx OI-Tx IO-Tx

Oﬀshore site Inshore site 20 km

FIGURE 2 (a) Map of the study area and (b) morphology of Tubastraea coccinea samples during the study. (a) To assist orientation, the chart marks positions of the offshore and inshore experimental sites and depicts the direction of transplantations. (b) Photographs showing the gross morphology of coral fragments. Key: O, offshore site; I, inshore site; IO-Tx, inshore to offshore transplant; OI-Tx, offshore to inshore transplant; T0, start of the experiment; T6, end of experiment (6 weeks)

The data-dependent mode was set to cycle time with 3 s between A second analysis was carried out to assign putative biological func- master scans. Precursors were filtered according to charge state (to tions to sequences not identified as potential toxins, by comparison include charge states 2–7), with monoisotopic precursor selection against the annotated transcriptome of Eguchipsammia fistula (Yum and using an intensity range of 5E3 to 1E20. Previously interrogated et al., 2017), the closest species to T. coccinea for which gene pre- precursors were excluded using a dynamic window (40 s ± 10 ppm). dictions are available. Sequences were assigned an annotation that The MS2 precursors were isolated with a quadrupole mass filter set gave a match against the E. fistula transcriptome with an e-value of to a width of 1.6m/z. ITMS2 spectra were collected with an AGC <1.0e–5. The PEAKS Q algorithm was used to measure the abun- target of 5,000, maximum injection time of 50 ms, and HCD collision dance of peptides across the TMT6plex dataset where TMT reporter energy of 35%. ion signals were above the limit of quantitation (LOQ value). The value for each peptide in a protein was summed into one measure- ment according to an up(+) or down(−) fold-change calculation. The 2.6 | Data analysis quantitative results were represented as a clustered heat map (dou- ble dendrogram) using Neighbor-joining tree clustering based on the PEAKS v8.5 software (Bioinformatics Solutions Inc.,) was used Euclidean distance. MS/MS spectra corresponding to regulated fea- for de novo sequencing, annotation, and quantification of unique tures of interest were manually reviewed to validate the assignment, MS/MS events (File S1 in the PRIDE repository dataset identi- and these raw spectral data have been deposited via the PRIDE part- fier PXD015559). Potential toxin sequences were annotated by ner repository with the dataset identifier PXD015559. homology searching against a custom toxin database comprising the UniProtKB/SwissProt-ToxProt dataset (Jungo, Bougueleret, Xenarios, & Poux, 2012) and previously published putative cnidar- 3 | RESULTS ian toxin proteins (Jaimes-Becerra et al., 2019). Protein sequences with an e-value of <1.0e–5 to entries in the custom toxin database There were no apparent gross morphological differences among were used as input to a machine learning tool called “ToxClassifier” the coral fragments collected from the offshore or inshore sites that excludes proteins with possible nontoxic physiological func- at T0 and T6; and the transplanted Tx fragments recovered at the tions (Gacesa, Barlow, & Long, 2016). Sequences that met the strin- two experimental sites. The coral skeleton and soft tissues formed gent validation process were considered bone fide potential toxins. typical clumps of pink-red calcareous cups around a single deep red 1798 | KITAHARA et al. or orange polyp, with yellow to bright orange tentacles (Figure 2). 24/74 (32.4%) of proteins shifted to levels of expression equiva- The composition and abundance of cnidae types were also uniform lent to the native T6 fragments collected at the offshore site. For between T0, T6, and Tx offshore and inshore fragments, consist- 30/74 (40.5%) of proteins in this Tx fragment, fold-changes did not ent with expected variation between specimens (Table 1). The most resemble the levels of protein expression in either of the local T6 common capsule types were spirocysts, tentacle type holotrichous coral fragments from offshore or inshore sites. and tentacle type b-rabdoids; while mesentery type holotrichous and mesentery type b-rabdoids were also noticed, but to a lesser extent. All morphological features are consistent for this species 4 | DISCUSSION (Picciani et al., 2011). The composition of recognized toxins extracted from specimens' This study examined potential differences in venom toxins of T. coc- nematocysts was identical irrespective of site, time of sampling, cinea colonies inhabiting an inshore and offshore site in southern and transplantation (File S1 PRIDE repository dataset identifier Brazil. The potential for T. coccinea venom to adapt its toxin com- PXD015559). The venom of T. coccinea is composed of 19 identi- position in response to different habitat environments was also as- fied protein toxins with predicted neurotoxic (8/19), cytolytic (6/19), sessed by manipulating a 6-week reciprocal transplant trial between or dyshomeostasis (5/19) toxicological functions (Table 2). No sig- the study sites. While there were some significant site-specific nificant effect of site or treatment was found on the fold-change expression differences in the global proteome of T. coccinea and expression of the 10 putative toxins which met the criteria for quan- its response to reciprocal transplantation over the 6-week study tification (proteins used for quantification are labeled in Table 2 and period (Figure 3 and File S2 PRIDE repository dataset identifier the quantification data are given in File S1(PRIDE repository dataset PXD015559), quantitative proteomic analyses clearly show that identifier PXD015559). there was no difference in venom toxin expression in populations An additional 74 proteins from the discharged nematocysts inhabiting the geographical confines of our study area. These results were annotated by molecular comparison to the published E. fis- are surprising because animal venoms are typically composed of tula transcriptome (Yum et al., 2017); these nontoxin proteins many constituent toxins that together manifest as the venom phe- were common in all samples and met the criterion for quantifi- notype. Hence, we can hypothesize that strong selection is acting cation (File S2 PRIDE repository dataset identifier PXD015559). upon these toxic constituents to refrain adaptation to possible dif- The average fold-change of peptides associated with each site and ferences in feeding and defense pressures across geographically and treatment is displayed as a heat map (Figure 3), in which values <2 environmentally separate habitats, although not necessarily distinct or >2 were considered either a significant reduction or increase T. coccinea populations. in protein tag intensity. Overall, the trends in nontoxin peptide Dichotomy in venom phenotypes has been extensively de- fold-changes were different at T0 and T6 (i.e., six weeks) between scribed on geographical and environmental scales in many ven- offshore and inshore coral fragments (File S2 PRIDE repository omous bilaterians. These patterns were initially deduced upon dataset identifier PXD015559). Corals at the offshore site shared observing dissimilar symptoms following envenomation occurring 50/74 (67.6%) of proteins with the same levels of expression be- from a common species. More recently, the hypothesis was tested tween the T0 and T6 specimens. Likewise, 41/74 (55.4%) of pro- by direct toxin analysis and from next-generation sequencing data teins did not have significant differences in expression between annotation (Zancolli et al., 2019). A classic example of venom poly- the T0 and T6 specimens at the inshore site. Hence, fold-change morphism in geographically distinct populations is the presence or comparisons were made between the T6 and Tx proteomes (i.e., absence of neurotoxic phospholipase A2 in Mojave Rattlesnakes at six weeks only). Transplantation between sites had a marked (Crotalus scutulatus) across the Sonoran Desert of the southwest- effect on protein fold-changes (Figure 3 and File S2 PRIDE re- ern United States and northern Mexico. The absence of the phos- pository dataset identifier PXD015559). After six weeks, the Tx pholipase confers a less potent hemorrhagic venom phenotype, fragment transplanted from the offshore to inshore site retained resulting in local and differential selection for a greater proportion 13/74 (17.6%) of proteins with similar levels of expression to of mammals over lizard prey items (Strickland et al., 2018). A previ- corresponding proteins in the native offshore T6 proteome. The ous study that compared the toxicological characteristics of cnidar- fold-changes in 30/74 (40.5%) of proteins in this Tx specimen sig- ians, however, concurred with our results. In that study (Radwan et nificantly shifted six weeks after transplantation to resemble lev- al., 2001), the cnidome toxin composition of Cassiopea and Aurelia els of expression to the same proteins in the T6 coral at the inshore species collected from very distinct habitats and geographical loca- site. Surprisingly, 31/74 (41.9%) of the Tx proteome had protein tions (viz. Bahamas, Chesapeake Bay, and Red Sea) were identical. fold-changes that were significantly different to the T6 specimen The toxicological profiles of the venoms from isolated nematocysts from the offshore to inshore sites. Protein fold-changes in the were also alike between locations for each species; nevertheless, nontoxin proteome of the Tx fragment relocated from inshore to the potency of the venom in the Red Sea specimens was much offshore sites was also affected six weeks after transplantation. greater (Radwan et al., 2001). While fold-changes in 20/74 (27.0%) of proteins were expressed After six weeks, fragments of native and transplanted colonies at identical levels as those of the T6 specimen at the inshore site, collected at the sample sites retained identical macro (skeletal and KITAHARA et al. | 1799

TABLE 1 Cnidome composition of Treatment Offshore Inshore Tubastraea coccinea specimens sampled offshore, inshore and those transplanted T0 Spirocyst—very common Spirocyst—very common between the two sites Tentacle type holotrichous—common Tentacle type holotrichous—common Tentacle type b-rhabdoid—common Tentacle type b-rhabdoid—common Mesentery type holotrichous—rare Mesentery type b-rhabdoid—rare T1 Spirocyst—very common Spirocyst—very common Tentacle type holotrichous—common Tentacle type holotrichous—common Tentacle type b-rhabdoid—common Tentacle type b-rhabdoid—common Mesentery type holotrichous—rare Mesentery type b-rhabdoid—rare Tx Spirocyst—very common Spirocyst—very common Tentacle type holotrichous—common Tentacle type holotrichous—common Tentacle type b-rhabdoid—common Tentacle type b-rhabdoid—common Mesentery type holotrichous—rare Mesentery type b-rhabdoid—rare

Note: The identification and abundance of different nematocyst types followed the scheme of Picciani et al. (2011).

TABLE 2 Predicted venom proteome of potential toxins from nematocysts of Tubastraea coccinea

Possible toxin Example of animal species with closest Toxin with closest homology function Accession numbers homology

a α-Latrotoxin-Lhe1a Neurotoxin Toxin3081 P0DJE3 Latrodectus hesperus (Western black widow spider) Calglandulin Neurotoxina Toxin6709 adi_v1.03437* Acropora digitifera (Staghorn coral) Calglandulin Neurotoxin Toxin6710 adi_v1.01102* Acropora digitifera (Staghorn coral) Basic phospholipase A2 Neurotoxina Toxin3317 O42187 Gloydius halys (Siberian pit viper) Basic phospholipase A2 Neurotoxin Toxin3579 P14556 Naja pallida (Red spitting cobra)

Ω-theraphotoxin-Hs1a Neurotoxin Toxin5919 P68424 Haplopelma schmidti (Chinese bird spider) Stonustoxin subunit-α Neurotoxin Toxin4963 Q98989 Synanceia horrida (Estuarine stonefish) + K channel toxin α-KTx 4.2 Neurotoxin Toxin2551 P56219 Tityus serrulatus (Brazilian yellow scorpion) Endothelin-converting enzyme 1 aCytolysin Toxin6732 Ponce et al. (2016) Chrysaora fuscescens (Pacific sea nettle) Gigantoxin-4 Cytolysina Toxin6744 H9CNF5 Stichodactyla gigantea (Giant carpet anemone) Waprin-Phi1 Cytolysina Toxin6649 A7X4K1 Philodryas olfersii (South American green snake) Phospholipase D Cytolysina Toxin0776 C0JB53 Sicarius peruensis (Six-eyed sand spider) Phospholipase D Cytolysin Toxin0293 C0JB21 Loxosceles rufescens (Recluse spider) Phosphodiesterase Cytolysina Toxin6676 adi_v1.12125* Acropora digitifera (Staghorn coral) Disintegrin Dyshomeostasisa Toxin6705 adi_v1.15751* Acropora digitifera (Staghorn coral) Disintegrin Dyshomeostasis Toxin6487 P18619 Protobothrops flavoviridis (Habu pit viper) Disintegrin Dyshomeostasis Toxin6701 adi_v1.17845* Acropora digitifera (Staghorn coral) Snaclec 7 Dyshomeostasis Toxin6686 adi_v1.12298* Acropora digitifera (Staghorn coral) Flavoxobin Dyshomeostasisa Toxin4271 P05620 Protobothrops flavoviridis (Habu pit viper)

Note: Putative toxins were annotated by homology of peptide sequences obtained from de novo sequencing of unique peptide MS/MS events with a custom database of known animal venom toxins. Venomous animals and their toxins with closest sequence similarity are given together with accession numbers corresponding to either UniProt or * ZoophyteBase (Dunlap et al., 2013) assignments. Note that the accession numbers in the left-hand column refer to the laboratory numbers used for the proteomics analysis given in File S1 (PRIDE repository dataset identifier PXD015559). aPotential toxins which met the criteria for quantification, data and calculations for which are given in File S1 (PRIDE repository with the dataset identifier PXD015559). soft tissue features, Figure 2) and micro (cnidome, Table 1) morphol- proteome of the 2016 specimen was also remarkably consistent with ogies, as well as venom compositions (Table 2). The putative toxin our findings presented herein, both in the number of constituent tox- proteome obtained from isolated tentacle nematocysts of T. coc- ins and their predicted toxicological activities: 8/16 neurotoxic, 2/16 cinea was recently reported for specimens collected from the São cytolytic and 6/16 dyshomeostatic toxins. Indeed, a similar obser- Sebastião Channel in 2016 (Jaimes-Becerra et al., 2019). The venom vation was reached by comparing the venom proteomes of Olindias 1800 | KITAHARA et al.

FIGURE 3 Heatmap showing quantified changes of proteins from discharged nematocysts isolated from Tubastraea coccinea specimens sampled offshore, inshore, and those transplanted between the two sites. Fold-change values were considered either a significant reduction (<2) or significant increase (>2) in tag intensity

sambaquiensis from specimens collected 5 years apart attesting that invariant venom, with both venom and the delivery system hav- cnidarian venom composition is stable over time, at least in popula- ing co-evolved independently of geographical or environmental tions of O. sambaquiensis from the São Sebastião Channel (Doonan forces. Such may reveal, however, that relative concentrations of et al., 2019). We are currently undertaking a 3-year noninterrupted certain toxins may vary between nematocyst types (Doonan et al., time-series experiment to monitor the venom toxin composition of 2019) or in species from different habitats, perhaps altering venom certain cnidarians in the São Sebastião Channel. potency by varying nematocyst types and densities to allow ad- A recent study presented quantitative proteomics that ac- aptation concerning to prey preference or predator type, which curately determined the composition and relative abundance of warrants further investigation. toxins present in enriched preparations of two nematocyst types It may be argued that the six-week study period was not long isolated from the primary tentacles of the adult medusa stage of enough for T. coccinea to adapt change in its venom composition, the hydrozoan O. sambaquiensis. The venom composition of the cnidome, and gross morphology following transplantation between two capsule types was nearly identical, and there was little dif- the offshore and inshore sites. T. coccinea is an azooxanthellate and ference also in the relative abundance of toxins between the two obligate heterotroph coral with an extreme regenerative capacity nematocyst preparations (Doonan et al., 2019). A pattern is emerg- (Luz et al., 2018) that depends on prey capture to meet its metabolic ing from published data (Doonan et al., 2019; Radwan et al., 2001) needs, including its reproductive energy demands. It was thus ex- and that of this study, whereby cnidarians appear to retain an array pected that the venom composition of T. coccinea would be different of different nematocyst types designed to deliver a single and in colonies sampled at inshore and offshore sites, due to potential KITAHARA et al. | 1801 disparities in habitat prey populations. Yet, there were significant 5 | CONCLUSIONS fold-changes in the cnida proteomes of specimens collected at each site and those following reciprocal transplantation after six weeks The complexity of toxins in isolated nematocysts from the hetero- of acclimation. Nevertheless, it should be noted that the natural trophic coral T. coccinea did not vary significantly in genotypically distributional range of this southwestern Atlantic invasive species identical colonies taken from offshore and inshore sites, or follow- has a much more diverse coral fauna and, therefore, complex “within ing reciprocal transplantation of clonal fragments between the two order” substrate competition. Such competition might have shaped sites. The structural morphology and cnidome toxin composition T. coccinea venon/toxins, which are potent enough to outcompete also did not change during the 6-week study period. These findings the native species from invaded areas. suggest that T. coccinea produces a single and relatively invariant However, comparatively, it is evident that a six-week period was venom, and that the venom and its delivery system are conservative sufficient for transcription and translation processes to alter expres- and expressed independently of geographical influence and environ- sion patterns in the nontoxin proteome of T. coccinea nematocytes, mental selection in the invaded habitats of the northern São Paulo but such adaptation certainly did not extend to the venom pheno- coast. type. Venomous anthozoans often elaborate the same combination and potency of toxins for both predation and defense, potentially ACKNOWLEDGMENTS where constitutive expression is adequate for both purposes, even We thank Prof Beth Okamura and Dr Alvaro Esteves Migotto for during a severe “bleaching” event causing an enhance requirement their advice during the early stages of this project and to Dr Walter for heterotrophic metabolism (Hoepner, Abbott, & Burke da Silva, C. Dunlap for his critical review of the manuscript. We are grateful 2019). Changes in predator exposure, but not diet, elicit a larger to the staff at CEBIMar for providing logistic support of shipboard venom proportion of defensive toxins than predatory toxins in the operations and onshore technical assistance. Financial support was scorpion Liocheles waigiensis (Gangur et al., 2017), and remarkably, provided from the UK by the Leverhulme Trust (grant number RPG- carnivorous cone snails are reported to switch between distinct 2016-037) and, from Brazil by FAPESP (grant numbers 2011/50242- venoms in response to predatory or defensive stimuli (Duterte et 5, 2014/01332-0), CNPq (grant numbers 309995/2017-5, al., 2014). The only known predators of T. coccinea in the tropical 301436/2018-5), CAPES (grant number 001) and the Universidade Indo-Pacific are the gastropod Epidendrium billeeanum (Rodríguez- de São Paulo (grant number 13.1.1502.9.8). This research study is a Villalobos, Ayala-Bocos, & Hernandez, 2016) and the nudibranch contribution to the NP-BioMar program at the Universidade de São Phyllida melanobrachia (Okuda, Klein, Kinnel, Li, & Scheuer, 1982), Paulo. while generalist predators are yet to be detected within invaded habitats of the southwestern Atlantic (Moreira & Creed, 2012). We CONFLICT OF INTERESTS are using time lapse photography in current studies to record pre- None declared. dation of T. coccinea by native carnivores within our Brazilian study sites with a view to test the venom response of T. coccinea on expo- AUTHOR CONTRIBUTIONS sure to sustained threats of predation. MVK and PFL designed research; MVK, AJB, EGM, MW, and PFL Although the offshore and inshore sites were spatially sepa- performed research; MW, GP, ACMa, and ACMo contributed ana- rated and environmentally distinct habitats, it was assumed that the lytical tools; and all authors analyzed data and wrote the paper. proximate composition of the pelagic community would be similar at both locations since epifaunal benthic populations were some- DATA AVAILABILITY STATEMENT how alike. Thus, the venom phenotype would be unchanged if pre- The mass spectrometry proteomics data have been deposited dation alone were selective and if abiotic factors were not driving to the ProteomeXchange Consortium via the PRIDE (Perez- toxin diversification, as well as if the potency of the venom toxins Riverol et al., 2018) partner repository with the dataset identifier is adequate across a broad spectrum of ecotypes. Changes in diet PXD015559. have been associated with divergence in venoms during the early life history of cnidarians. For example, Underwood and Seymour ORCID (2007) have documented distinct differences in tentacle venom Marcelo V. Kitahara https://orcid.org/0000-0003-4011-016X protein profiles using 1D SDS–PAGE analyses between juvenile and Paul F. Long https://orcid.org/0000-0001-6698-4602 mature Carukia barnesi medusae (the highly toxic Australian Irukandji jellyfish). Likewise, Columbus-Shenkar et al. 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